DESCRIPTION: The global objective of the research proposed here is to understand the fundamental question of how a small RNA enzyme, called the hammerhead ribozyme, works. Using a combination of conventional and time-resolved X-ray crystallography experiments, together with synthetically modified RNA, the hypothesis that the recently elucidated ground-state structure of the hammerhead ribozyme is catalytically relevant will be tested. In doing so, an understanding of the relationship between catalytic RNA structure and function will be obtained by investigating the structures of the hammerhead ribozyme at various important points along the RNA cleavage reaction coordinate. The specific aims of the program described in this research proposal are: (1) to capture the crystal structures of hammerhead RNA cleavage intermediates; (2) to obtain the crystal structure of the hammerhead RNA after cleavage; (3) to capture structures of rate-limiting species during ligation of the cleaved hammerhead ribozyme substrate; (4) to obtain the structure of a hammerhead RNA cleavage reaction transition-state analog; (5) to understand the structural basis of catalysis-enhancing hammerhead RNA modifications; and (6) to select for a protein that binds to, and locks, the hammerhead RNA in a catalytically active conformation, and to determine the crystal structure of this RNA-protein complex. These six specific aims are each designed to probe, independently, the cleavage mechanism of this small RNA enzyme. The potential use of hammerhead ribozymes as therapeutic agents that target RNA viruses (such as HIV) and pathological mRNAs (such as oncogene transcripts) is currently an active area of research in the medical community. Hammerhead ribozymes as potential pharmaceuticals are of particular interest because they may prove usful for treating conditions that have proven otherwise intractable. One of the primary motives for the research proposed here, in addition to answering questions of a basic scientific nature, is to provide practical information to the scientific and medical communities to enable more potent ribosome-based pharmaceuticals to be designed.